Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans Akihiro Morita 1 , Haruhiko Isawa 2 , Yuki Orito 1 , Shiroh Iwanaga 3 , Yasuo Chinzei 1 and Masao Yuda 1 1 Department of Medical Zoology, School of Medicine, Mie University, Edobashi, Tsu, Japan 2 Department of Medical Entomology, National Institute of Infectious Diseases, Toyama, Sinjyuku-ku, Tokyo, Japan 3 Laboratory of Chemistry and Utilization of Animal Resources, Faculty of Agriculture, Kobe University, Hyogo, Japan Injury to the blood vessel wall exposes the subendo- thelial extracellular matrix, which is rich in collagens, providing a substrate for platelet adhesion and aggre- gation. This is the first step of hemostasis ending in formation of the thrombus. Several proteins on the platelet membrane participate in collagen–platelet interactions in a direct or indirect manner [1]. Glyco- protein (GP)VI [2–5] and a 2 b 1 integrin [6] bind to col- lagen directly, and GPIb-V-IX [7] and a IIb b 3 integrin [6,8] bind via von Willebrand factor (vWF). In the cur- rent ‘two-site, two-step’ model of platelet–collagen interaction [9–12], platelet aggregation proceeds as fol- lows: first, GPIb-IX-V rapidly binds to vWF immobi- lized on collagen so that passing platelets are tethered to the latter. Following this event, GPVI, a surface signaling receptor, binds to collagen with low affinity, which triggers the signaling cascade for platelet activa- tion. This leads to ‘inside-out’ activation of a 2 b 1 and a IIb b 3 integrins and secretion of platelet agonists such as ADP and thromboxane A 2 , accelerating platelet aggregation and thrombus formation. Therefore, GPVI has a central role in the initial phase of thrombus for- mation as the major signaling receptor for collagen. GPVI is composed of two extracellular immunoglob- ulin domains, a mucin-rich stalk, a single transmem- brane domain, and a short cytoplasmic tail [2–4]. GPVI is coupled to the Fc receptor (FcR) c-chain homodimer in the transmembrane domain via a salt bridge [5,9,10,13,14]. The binding of collagen to GPVI leads to cross-linking of GPVI molecules [15], inducing tyrosine phosphorylation of the cytoplasmic tail of FcR c-chains. This phosphorylation leads to binding Keywords collagen-induced platelet aggregation inhibitor; triplatin; Triatoma infestans Correspondence M. Yuda, Department of Medical Zoology, School of Medicine, Mie University, Edobashi, Tsu, Mie 514–8507, Japan Fax: +81 59231 5215 Tel: +81 59231 5013 E-mail: m-yuda@doc.medic.mie-u.ac.jp (Received 1 March 2006, revised 19 April 2006, accepted 4 May 2006) doi:10.1111/j.1742-4658.2006.05306.x To facilitate feeding, certain hematophagous invertebrates possess inhibi- tors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Tria- toma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major colla- gen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor c-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that trip- latins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates. Abbreviations CRP, collagen-related peptide; FcR, Fc receptor; GP, glycoprotein; MBP, maltose-binding protein; PGE 1 , prostaglandin E 1 ; PRP, platelet-rich plasma; vWF, von Willebrand factor. FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS 2955 and subsequent activation of the tyrosine kinase, Syk, which initiates downstream signaling events [16]. Various platelet inhibitors are found in hematopha- gus invertebrates [17,18]. These inhibitors interfere with platelet aggregation in host animals and facilitate blood feeding. They include inhibitors of collagen-sti- mulated platelet aggregation, but only a small number of inhibitors of this type have been characterized. LAPP [19–21] and calin [22] were identified from Haementeria officinalis and Hirudo medicinalis, respect- ively. They prevent both the binding to collagen of a 2 b 1 and the binding of GPIb-IX-V to vWF, inhibiting platelet aggregation and platelet adhesion to collagen. Moubatin [23,24] and pallidipin [25] were identified from Ornitohdoros moubata and Triatoma pallidipennis, respectively. These proteins inhibit platelet aggregation induced by collagen, but not platelet adhesion to colla- gen under static conditions. However their inhibitory mechanisms and target molecules remain unknown. Here, we have identified the novel platelet aggrega- tion inhibitors triplatin-1 and -2, from T. infestans. They share sequence similarity with pallidipin and spe- cifically inhibit platelet aggregation induced by colla- gen. We suggest that triplatins target GPVI and block platelet activation induced by collagen. Results Cloning and production of triplatin-1 and -2 recombinant proteins A cDNA library was constructed from the salivary glands of unfed T. infestans. Five hundred and fifty clones were randomly picked from this library and sequenced. Among them, the most abundant species (25 clones) corresponded to a cDNA encoding a 178 amino acid protein with a molecular mass of 19.5 kDa. In addition, an isoprotein of this abundant clone (four additional clones) was also found. This iso- form contains 182 amino acids with a molecular mass of 19.8 kDa. Both molecules were predicted to be secretory proteins by the signal p program, and both shared sequence similarities with pallidipin, a platelet inhibitor found in T. pallidipenis (Fig. 1). Identities of the abundant clone and its isoprotein with pallidipin are 63 and 49%, respectively. We named this salivary gland protein and its isoprotein ‘triplatin-2’ and ‘trip- latin-1’, respectively. To investigate the function of these triplatins, recombinant proteins were produced in a baculovirus- insect cell system. Secreted recombinant proteins formed a major fraction of all the proteins in the cell culture medium. They were purified by cation- exchange and gel filtration chromatography. Purity was confirmed by SDS ⁄ PAGE (Fig. 2). Apparent molecular masses of purified recombinant triplatin-1 and -2 were approximately 17 kDa on SDS ⁄ PAGE, which agreed with their predicted molecular masses of 17.9 and 17.1 kDa in secreted form. To identify native proteins in the saliva, antisera were raised against recombinant triplatin-1. In western blot analysis, the sera reacted with both triplatin-1 and -2, although relatively weakly with the latter (Fig. 2). The sera also reacted with native proteins in salivary glands, showing that both triplatin-1 and -2 are indeed expressed there. SDS ⁄ PAGE and western blot analysis indicated that triplatin-1 and -2 are major proteins of T. infestans saliva. Fig. 1. Alignment of triplatin-1 and -2 sequences. Multiple align- ment of triplatin-1 and -2 sequences with pallidipin from T. pallidi- pennis by CLUSTAL W. Underlining, asterisks, colons and periods indicate signal sequences, single fully conserved, strongly con- served and weakly conserved residues, respectively. Fig. 2. Western blot analysis of triplatin-1 and -2 from the salivary gland of T. infestans. Extracts from a pair of salivary glands, recom- binant triplatin-1 and recombinant triplatin-2 were separated by 15% SDS ⁄ PAGE under reducing conditions and stained by Coo- massie brilliant blue and immunoblotted with anti-triplatin-1 serum (a-triplatin-1). Identification and characterization of triplatin A. Morita et al. 2956 FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS Inhibition of platelet aggregation by triplatin-1 and -2 To investigate the function of triplatin-1 and -2, their effects on platelet aggregation were tested using plate- let-rich plasma (PRP) and platelet agonists, because they shared similarities with the known platelet aggrega- tion inhibitor, pallidipin, as described above. As shown in Fig. 3, triplatin-1 and -2 did not inhibit ADP- and thrombin-induced platelet aggregation, but slightly inhibited that induced by arachidonic acid and U46619, a thromboxane A 2 analog. However, this weak inhibi- tion did not titrate in a concentration-dependent man- ner, suggesting that it was an artifact. In contrast, both triplatin-1 and -2 clearly inhibited collagen-induced aggregation in a dose-dependent manner. Maximum inhibition of 50–60% at micromolar concentrations of triplatin-1 and -2 were recorded. These results demon- strated that triplatin-1 and -2 are inhibitors of platelet aggregation induced by collagen. To investigate precisely the effect of triplatin-1 and -2 on platelet aggregation caused by collagen, an inhi- bition assay was performed using washed platelets instead of PRP. In this assay, both triplatin-1 and -2 completely inhibited platelet aggregation induced by collagen (Fig. 4A). Based on these results, IC 50 values of triplatin-1 and -2 were calculated at approximately 60 nm and 620 nm, respectively. We also examined inhibition of collagen-induced platelet aggregation using an aggregometer and PRP (Fig. 4B). After sti- mulation by collagen, the characteristic peak caused by a change of platelet shape was observed within a few minutes. Triplatin-1 completely inhibited collagen- induced platelet aggregation and also attenuated the change of platelet shape. Identification of the target receptor for triplatin-1 and -2 Next, we attempted to identify triplatin-1 and -2 target molecules. Collagen-induced platelet aggregation is initiated by the interaction between vWF and GPIb-IX- V. Ristocetin promotes this interaction and aggregates platelets [9–12]. Therefore, we first examined the effects Fig. 3. Effect of triplatin on platelet aggregation induced by several different agonists. Platelets in platelet rich plasma (PRP) (3.5–3.6 · 10 5 plateletsÆmL )1 ) were incubated with triplatin at 37 °C for 10 min before adding collagen (2.0 l gÆmL )1 ), ADP (0.5 l M), arachidonic acid (1.0 m M) or U46619 (2.0 lM). For stimulation by 0.1 nM thrombin, washed platelets (2.6 · 10 5 plateletsÆmL )1 ) were used instead of PRP. Light transmittance at 600 nm was measured 10 min after stimulation, and platelet aggregation is represented as the percentage of control aggregation of platelets preincubated in the absence of triplatin. Results are the mean ± SE of three experiments. A. Morita et al. Identification and characterization of triplatin FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS 2957 of triplatin-1 and -2 on ristocetin-induced platelet aggre- gation (Fig. 4A). No inhibitory effect on platelet aggre- gation induced by ristocetin was observed, indicating that GPIb-IX-V is not a target for triplatin. Collagen-related peptide (CRP) contains the specific GPVI recognition motif and directly activates this receptor [26–28]. Therefore, we next examined whether triplatin-1 and -2 inhibit CRP-induced platelet aggrega- tion. Triplatin-1 and -2 did inhibit CRP-induced plate- let aggregation in a dose-dependent manner (Fig. 4A). We also examined inhibition of collagen-induced plate- let aggregation using an aggregometer (Fig. 4B). Trip- latin-1 almost completely inhibited CRP-induced platelet aggregation and also attenuated the change of platelet shape. These results demonstrated that triplat- ins inhibit platelet activation mediated by GPVI. We also assessed the effect of triplatin-1 and -2 on platelet adhesion to immobilized soluble collagen (Fig. 5), which has been reported to be entirely dependent on interactions between collagen and a 2 b 1 integrin. In control experiments, monoclonal antibody against integrin a 2 b 1 , Gi9, did inhibit platelet adhesion. However, triplatins did not inhibit platelet adhesion, even at a concentration of 1.0 lm. These results indicate that triplatins are specific inhibitors of collagen-induced platelet aggregation mediated by GPVI. To confirm this finding, we exam- ined the effect of triplatin-1 on phosphorylation of the FcR c-chain [5,13,14]. This was achieved by immuno- precipitation, because the binding of GPVI to collagen causes tyrosine phosphorylation of the FcR c-chain, which is coupled to GPVI. In the absence of triplatin- 1, phosphorylated FcR c-chain of platelets stimulated by 10 lgÆmL )1 collagen was clearly detected (Fig. 6). In contrast, in the presence of 1.0 lm triplatin-1, phos- phorylation of FcR c-chain was completely absent. Discussion GPVI is a surface signaling receptor on platelets and has a central role in platelet activation by collagen [10]. Here, we have identified a novel type of platelet Fig. 4. (A) Effect of triplatin on platelet aggregation induced by agonists of collagen receptors. Platelets in PRP (3.4 · 10 5 plateletsÆmL )1 ) incubated with triplatin were stimulated 1.25 mgÆmL )1 ristocetin. Washed platelets (3.3–3.4 · 10 5 plateletsÆmL )1 ) incubated with triplatin-1 were stimulated with 2.0 lgÆmL )1 collagen or 0.25 lgÆmL )1 CRP. Further methods are as in Fig. 3 legend. (B) Anti-aggregatory properties of triplatin on collagen- or CRP-induced platelet aggregation. Washed platelets (3.0 · 10 5 plateletsÆmL )1 ) incubated with triplatin-1 at 37 °C for 2 min were induced to aggregate by 2.0 lgÆmL )1 of collagen or 0.2 lgÆmL )1 of CRP. After stimulation, light transmittance was monitored by aggregometer for 10 min. Identification and characterization of triplatin A. Morita et al. 2958 FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS aggregation inhibitor, triplatin-1 and its isoform, trip- latin-2, which specifically inhibit platelet aggregation induced by collagen, especially by CRP, a specific agonist of GPVI [26–28]. Furthermore, triplatin-1 inhibited tyrosine phosphorylation of FcR c-chains induced by collagen, the initial step in the GPVI signa- ling cascade [13,14]. These results strongly suggest that triplatin-1 and -2 are antagonists for GPVI. To the best of our knowledge, triplatin-1 and -2 are the first natural inhibitors for GPVI to be identified. The injured arterial wall exposes collagen to the blood and recruits platelets to the injured site. In the physiological state, in which shearing has an important role, GPVI is involved in recruitment and subsequent aggregation of platelets. It was reported that platelets from GPVI-deficient mice show no adhesion to colla- gen and no aggregation [9]. In humans, platelets from GPVI-deficient patients can attach to collagen but nonetheless do not form aggregates. These findings suggest that GPVI is crucial for thrombus formation after arterial injury [13]. Feeding activities of hemato- phagous arthropods injure the blood vessels of host animals. It is likely that triplatin-1 and -2 are injected into the host during T. infestans blood feeding and attenuate host hemostasis at the initial phase. To date, some other inhibitors of collagen-induced platelet aggregation have been reported in the saliva of blood-sucking arthropods. Among them, pallidipin and moubatin are similar to triplatin in their inhibitory properties. Like triplatin, pallidipin and moubatin inhi- bit platelet aggregation induced by collagen but not by other agonists [25]. They also exert potent inhibitory effects on both platelets in plasma and washed plate- lets. Furthermore, unlike other inhibitors of collagen- induced platelet aggregation, such as LAPP [19–21] and Calin [22], they do not inhibit the platelet adhe- sion to collagen mediated by a 2 b 1 integrin under static conditions [23,24]. Although their mechanisms of action are not fully understood, it is possible that pal- lidipin and moubatin are also GPVI antagonists. In summary, we have identified inhibitors of colla- gen-induced platelet aggregation from the saliva of T. infestans. Platelet–collagen interactions have an important role in thrombus formation, and GPVI plays a pivotal role therein. Further investigations on the inhibitory mechanisms of these insect proteins might lead to development of antiplatelet agents that antagonize thrombus formation at the initial phase. Experimental procedures Materials Prostaglandin E 1 , deoxycholate, sodium orthovanadate and genenase were purchased from Biogenesis Ltd. (Poole, UK), Nakarai Tesque, Inc. (Kyoto, Japan), ICN Biomedi- cals, Inc. (Aurora, OH, USA) and New England Biolabs, Inc. (Beverly, MA, USA), respectively. Apyrase, phenyl- methylsulfonyl fluoride, leupeptin and aprotinin were pur- chased from Wako Pure Chemical, Ind. Ltd. (Osaka, Japan). Collagen, ADP and ristocetin were purchased from Chronolog, Corp. (Havertown, PA, USA). U46619, thrombin, fibrinogen and Gly-Pro-Arg-Pro peptide were Fig. 5. Effect of triplatin on platelet adhesion to collagen. 3.0 · 10 5 washed platelets ⁄ well were incubated with triplatin-1, -2 or mono- clonal antibody against integrin a 2 b 1 (Gi9) for 10 min and introduced into wells coated with 2.0 lgÆwell )1 collagen. After washing, the adherent platelets were quantified using a protein assay. The relat- ive number of adherent platelets is presented. Results are the mean ± SE of three experiments. Fig. 6. Inhibition of the phosphorylation of FcR c-chain by triplatin. Anti FcR c-chain immunoprecipitates were prepared from lysates of platelets stimulated by 10 lgÆmL )1 collagen (10 lgÆmL )1 ) in the pres- ence or absence of 1.0 l M triplatin-1 and analyzed by anti-FcR c-chain (aFcR c-chain) and antiphosphotyrosine (aPY) immunoblotting. A. Morita et al. Identification and characterization of triplatin FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS 2959 purchased from Calbiochem (San Diego, CA). CRP was a kind gift of H. Takayama of Kyoto University. Arachido- nic acid, Nonident P-40, and other chemicals were pur- chased from Sigma-Aldrich, Inc. (St. Louis, MD, USA). Isolation and sequencing of cDNA clones Salivary glands of T. infestans were dissected from thoraces of unfed adults, and poly A(+) RNA was isolated from 30 pairs of salivary glands using a MicroPrep mRNA isolation kit (Amersham Pharmacia Biotech, Ltd, Amersham, Buck- inghamshire, UK). The salivary gland cDNA library was constructed from this isolated mRNA using the SuperScript plasmid system (Gibco BRL Life Technologies, Inc., Rock- ville, MD, USA) according to the manufacturer’s instruc- tions. From this constructed cDNA library, 550 clones were randomly selected and their DNA sequences were determined using ABI PRISM BigDye Terminator cycle sequencing kits (Applied Biosystems, Foster City, CA, USA), by ABI 310 genetic analyzer (Applied Biosystems). The cDNA sequences of triplatin-1 and -2 were deposited in the DNA Data Bank of Japan (DDBJ) (accession num- ber, AB 250209 and AB 250210). Sequence similarities and signal peptide prediction of these clones were carried out using blast (http://www.blast.genome.ad.jp/) and signal p programs (http://www.cbs.dtu.dk/services/SignalP/), respect- ively. Sequence alignment was performed using the clustal w program (http://www.clustal.genome.ad.jp/). Production and purification of the recombinant protein Triplatin-1 and -2 recombinant proteins were produced in a baculovirus-insect cell system. Full-length cDNA of tripla- tin-1 and -2 were cloned into the BamHI site of the baculo- virus transfer vector, pAcYM1. The Sf9 cells were cotransfected with the constructed plasmid and linearized baculovirus DNA, AcRp23, lacZ (Becton Dickinson Bio- sciences, San Jose, CA, USA). Recombinant proteins from Tn5 cells infected by the recombinant baculoviruses were secreted into the culture medium due to their original signal peptide sequences. They were purified by a combination of cation-exchange chromatography and gel-filtration chroma- tography. Briefly, culture supernatants containing secreted recombinant proteins were first applied to a PD-10 column (Amersham Pharmacia Biotech) in 20 mm sodium acetate buffer, pH 5.2. Next, the eluted samples were applied to a MONO S column (Amersham Pharmacia Biotech) equili- brated with 20 mm sodium acetate buffer, pH 5.2 and eluted with a gradient from 0 to 1 m NaCl at a flow rate of 1mLÆmin )1 . The fractions containing recombinant proteins were pooled, concentrated by Centricon 10 (Millipore Corp., Bedford, MA, USA) and applied to a TSK G2000 SW column (Tosoh, Tokyo, Japan) equilibrated with 50 mm Tris ⁄ HCl, pH 7.4, containing 150 mm NaCl. Purity of recombinant product was established by 15.0% SDS ⁄ PAGE under reducing conditions. Protein concentra- tion was determined with a Coomassie protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) using bovine c-globulin as the standard. Preparation of antibody to triplatin-1 To obtain a large amount of triplatin-1 to use as an immu- nogen, recombinant triplatin-1 was also produced as a maltose-binding protein (MBP)-fusion protein. Briefly, a cDNA fragment encoding triplatin-1 without signal peptide was amplified by PCR and cloned into an expression plas- mid, pMAL-c2q (New England Biolabs). The MBP-fused triplatin was produced using this plasmid in Escherichia coli BL21, and was purified by amylose-resin affinity chroma- tography. Cleavage of the recombinant protein from MBP was achieved using genenase. For antibody production, rats were immunized with the MBP-free triplatin-1 emulsified in TiterMax gold (CytRx Corp., Los Angeles, CA, USA). After the first immunization, booster immunizations were done twice at intervals of 2 weeks. After the final immun- ization, whole blood was taken and antisera against tripla- tin-1 were prepared. Western blot analysis Salivary gland proteins were separated by SDS ⁄ PAGE in 5–20% gradient gels and transferred to nitrocellulose mem- branes. Blotted membranes were blocked in NaCl ⁄ P i con- taining 5% skimmed milk, and incubated with primary antibody against triplatin-1, and then with alkaline phos- phatase-conjugated antirabbit IgG. The signal was detected using nitroblue tetrazolium. To detect the FcR c-chain in platelets, western blot analysis was performed according to Tulasne et al. [29]. Platelet proteins were separated by 4–20% gradient SDS ⁄ PAGE, and transferred to a polyvinylidene difluoride membrane. The FcR c-chain was detected with specific antisera (Upstate Biotechnology, Lake Placid, NY, USA) in a manner similar to that described above. Tyrosine phosphorylation was detected by monoclonal antibody 4G10 (Upstate Biotechnology). The signal was detected using an enhanced chemiluminescence detection system (SuperSignal West Pico Chemiluminescent Substrate, Pierce Biotechnology). Platelet preparation Blood was collected from healthy human volunteers by veni- puncture on acid–citrate–dextrose anticoagulant. PRP was obtained by centrifugation at 110 g for 10 min. Platelets were obtained by centrifugation at 1100 g for 20 min, fol- lowing incubation for 10 min with 20 ngÆmL )1 prostaglandin Identification and characterization of triplatin A. Morita et al. 2960 FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS E 1 (PGE 1 ), and washed three times using modified Tyrode’s buffer (5 mm Hepes buffer, pH 7.4, 134 mm NaCl, 3 mm KCl, 0.3 mm NaH 2 PO 4 ,2mm MgCl 2 ,5mm glucose, 12 mm NaHCO 3 ,1mm EGTA, 3.5 mgÆmL )1 bovine serum albu- min, 20 ngÆmL )1 PGE 1 ,20ngÆmL )1 apyrase). Finally, washed platelets were resuspended in modified Tyrode’s buf- fer, substituting 2 mm CaCl 2 for 1 mm EGTA. The number of platelets was counted using a hematocytometer. Effect of triplatin-1 and -2 on platelet aggregation and adhesion Effects of triplatin-1 and -2 on platelet aggregation were photometrically measured according to Bednar et al. [30]. PRP or washed platelets were incubated with triplatin-1 and -2 for 10 min in a 96-well flat-bottom plate (MICROTEST TM 96, Becton Dickinson Biosciences) in 50 mm Tris ⁄ HCl, pH 7.4, containing 150 mm NaCl. Plate- let aggregation was initiated by addition of 2.0 lgÆmL )1 collagen, 0.5 lm ADP, 1.0 mm arachidonic acid, 0.2 lm U46619, 0.1 nm thrombin, 1.2 mgÆmL )1 ristocetin or 0.25 lgÆmL )1 CRP as platelet activators. The reaction mixture was stirred continuously at 37 °C for 10 min. Platelet aggregation was monitored by light transmittance using a microplate reader (MPR-A4i, Tosoh, Tokyo, Japan). Effects of triplatin-1 and -2 on platelet aggregation induced by collagen and CRP were also analyzed using an aggregometer (C-500, Chronolog). PRP was mixed with triplatin-1 or -2, and platelet aggregation was started by addition of either 2.0 lgÆmL )1 collagen or 0.2 lgÆmL )1 CRP. Platelet aggregation was monitored by light transmit- tance in the aggregometer with continuous stirring at 37 °C. Inhibition of platelet adhesion was examined according to Keller et al. [19]. Microtiter plates (MicrotiterÒ Poly- styrene Base Immunoassay Plates, DYNEX Technologies, Inc., Chantilly, VA, USA) were coated with collagen (2.0 lgÆwell )1 )in5mm acetic acid for 1 h at room tem- perature, followed by addition of 1% bovine serum albu- min for 1 h at room temperature to block the nonspecific binding of platelets to the wells. After blocking, wells were washed three times with Hepes-buffered saline, 20 mm Hepes, pH 7.4 containing 0.14 m NaCl and 2 mm MgCl 2 . Washed platelets (3.0 · 10 5 cells) and triplatin or Gi9 (Immunotech, Marseille, France) were mixed in Tyrode’s buffer containing 2 mm CaCl 2 and 100 ngÆmL )1 PGE 1 and then transferred into wells. After 45 min incu- bation, wells were washed three times with Hepes-buf- fered saline again. The number of platelets adhering to immobilized collagen was determined using Micro BCA Protein Assay Kits (Pierce Biotechnology). The percentage of specifically adherent platelets was calculated on the basis of a standard curve obtained with known numbers of platelets. Platelet lysis and immunoprecipitation of FcR c-chains Immunoprecipitation was performed according to Ichinohe et al. [16] and Tulasne et al. [29]. Washed platelets (1 · 10 9 cells) were incubated with 1.0 lm triplatin-1 at 37 °C for 10 min. After incubation, platelets were stimulated by 10 lgÆmL )1 collagen at 37 °C for 10 min and then lyzed with an equal volume of ice-cold lysis buffer, 20 mm Tris, 300 mm NaCl, 2 mm EDTA, 2% Nonident P-40, 1% deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluor- ide, 2 mm sodium orthovanadate, 10 lgÆmL )1 leupeptin and 10 lgÆmL )1 aprotinin. Non-lysed cells and debris were removed by centrifugation. Platelet lysate was incubated with 1 mgÆmL )1 anti-FcR c-chain antiserum at 4 °C. After overnight incubation, protein A-Sepharose beads were added to the mixture and washed three times with 10 mm Tris, 160 mm NaCl and 0.1% Tween 20. The protein bound to beads was eluted with Laemmli buffer and applied to SDS ⁄ PAGE and western blot analysis. Acknowledgements We thank H. Takayama of Kyoto University for the generous gift of CRP. This study was supported by a grant-in-aid for Scientific Research (13006374) to HI, for Scientific Research on Priority Areas (08281103) to YC, for Scientific Research (B) (12470060) to MY, and for Exploratory Research (10877043, 11877043 and 12877042) to YC from the Ministry of Education, Sci- ence, Culture and Sports of Japan. 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